FLUORESCENCE STUDY OF TRP IN CHEY AFTER T-JUMP

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose to study the complex of CheY with a 16 residue peptide corresponding to the CheY binding domain FliM using tryptophan fluorescence and T-jump technology. The affinity between the peptide residue and CheY is weak when CheY is unphosphorylated and about 25 times stronger in the phosphorylated state. The intrinsic tryptophan fluorescence of the single Trp was found to be quenched on peptide binding, a signal loss that could be exploited to monitor binding in a kinetic experiment. Using NMR previously, a suggested mechanism may involve an isomerization between a predominant inactive conformation and a minority active conformation that is able to bind the peptide. In this view, the role of the phosphorylation is to shift the equilibrium between the active and inactive forms. We propose to monitor the binding kinetics using the laser T-jump technique in both resting and phosphorylated states. We are also proposing to investigate a collection of mutants with altered equilibria between the active and inactive conformation.